Patricia Roche

Intracellular signaling of cardiac fibroblasts.

Long regarded as a mere accessory cell for the cardiomyocyte, the cardiac fibroblast is now recognized as a critical determinant of cardiac function in health and disease. A recent renaissance in fibroblast-centered research has fostered a better understanding than ever before of the biology of fibroblasts and their contractile counterparts, myofibroblasts. While advanced methodological approaches, including transgenics, lineage fate mapping, and improved cell marker identification have helped to facilitate this new work, the primary driver is arguably the contribution of myofibroblasts to cardiac pathophysiology including fibrosis and arrhythmogenesis. Fibrosis is a natural sequel to numerous common cardiac pathologies including myocardial infarction and hypertension, and typically exacerbates cardiovascular disease and progression to heart failure, yet no therapies currently exist to specifically target fibrosis. The regulatory processes and intracellular signaling pathways governing fibroblast and myofibroblast behavior thus represent important points of inquiry for the development of antifibrotic treatments. While steady progress is being made in uncovering the signaling pathways specific for cardiac fibroblast function (including proliferation, phenotype conversion, and matrix synthesis), much of what is currently known of fibroblast signaling mechanisms is derived from noncardiac fibroblast populations. Given the heterogeneity of fibroblasts across tissues, this dearth of information further underscores the need for progress in cardiac fibroblast biological research.

TGFβ1 regulates scleraxis expression in primary cardiac myofibroblasts by a Smad-independent mechanism.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

Role of scleraxis in mechanical stretch-mediated regulation of cardiac myofibroblast phenotype.

The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.

Transcriptional control of collagen I gene expression.

Cardiac fibrosis is the pathological remodeling of the extracellular matrix (ECM) in response to stresses such as pressure overload or injury. While initially adaptive, myocardial remodeling and subsequent fibrosis causes increased wall stiffness, arrhythmias, cardiac dysfunction, and eventually heart failure. Though the disease processes and origins may differ, excess deposition of fibrillar collagens type I and III characterizes fibrosis in the heart, lungs, kidneys, liver, and skin. Under normal physiological conditions, high tensile strength collagen fibers maintain cardiac structural integrity, connect individual cardiomyocytes, transmit contractile force, and resist deformation and rupture of the ventricle during systole. Various factors contribute to the development of fibrosis by altering expression of ECM genes, including increased synthesis of pro-inflammatory cytokines, alterations in the levels of circulating hormones, and mechanical strain resulting from ECM degradation. This review focuses on the transcriptional mechanisms governing expression of the major cardiac collagen, type I. Key cis- and trans-acting regulators of collagen I gene expression are discussed. Surprisingly, relatively few transcriptional regulators of collagen synthesis have been identified specifically in cardiac fibroblasts. However, key players have been identified in other tissue and cell types, and are important to consider in elucidating the molecular mechanisms underpinning collagen gene expression in the heart in both health and disease.

Molecular Mechanisms of Cardiac Development

The heart is the first organ to develop in order to supply the ever-increasing metabolic demands of the growing embryo. The heart is a unique structure in the body as it is derived from four distinct pools of progenitors: the first heart field (cardiac crescent), the second heart field (SHF), the proepicardial organ and the cardiac neural crest. These progenitors differentiate into the different cell types that comprise the adult heart: cardiomyocytes, endothelial cells, vascular smooth muscle cells, fibroblasts, and the conduction system. This complex program of differentiation is controlled by different molecular signaling pathways. A key component of the cardiac development program is the exquisitely coordinated expression of various genes in a spatially and temporally controlled fashion. Genes must be activated or repressed within restricted regions at specific times in order for normal cardiac development to proceed. In large part, this regulation of gene expression is controlled by an evolutionarily conserved set of transcription factors and microRNAs (miRNAs). Historically, the study of cardiac transcription factors has been very informative in understanding the early events in cardiogenesis. The rapidly evolving field of cardiac miRNAs promises to further extend our understanding of cardiac development. In this chapter, we will describe essential cardiac transcription factors and miRNAs and their role in controlling cardiac development.

Pirfenidone and the Inflammasome: Getting to the Heart of Cardiac Remodeling

and cardiac fibrosis, including the development of fibrotic foci, fibroblast-to-myofibroblast conversion, and the presence of a chronic inflammatory state. By targeting these commonalities, pirfenidone may be effective in both diseases. Pirfenidone was initially reported to reduce fibrosis and improve lung function in a hamster model of bleomycin-induced lung injury [3, 4] . Now marketed as Esbriet ® in Europe and Canada, it is undergoing phase III trials in the US for treatment of mild/moderate IPF. By ameliorating lung function decline, pirfenidone appears to slow disease progression although additional data are needed regarding its efficacy in improving patient quality of life [5–8] . Recent studies reported beneficial effects of pirfenidone in various animal models of cardiac dysfunction, including diabetes, pacing-induced heart failure, Duchenne muscular dystrophy, doxorubicin cardiotoxicity, DOCA salt hypertension, and myocardial infarction [9– 14] . However, the means by which pirfenidone exerted these salutary effects is unclear, and improved cardiac function was usually but not always accompanied by reduced fibrosis, suggesting several modes of action. Clearly, more data are required to determine both the mechanism of action of pirfenidone and, more broadly, its applicability. In vitro studies demonstrate that pirfenidone Fibrosis is the progressive accumulation of interstitial matrix, resulting in impaired organ function and increased patient morbidity and mortality. The underlying mechanisms are often poorly understood even when the clinical progression of the disease is familiar. Effective treatments for fibrosis remain elusive due in part to the involvement of pleiotropic pathways that are challenging to target therapeutically, although hope remains for a ‘magic bullet’ capable of attenuating fibrosis regardless of the tissue. In this issue of Cardiology , Wang et al. [1] demonstrate that the anti-inflammatory small-molecule drug pirfenidone, currently used for idiopathic pulmonary fibrosis (IPF), may be efficacious for the treatment of cardiac fibrosis. Cardiac remodeling and fibrosis follow acute or chronic insults, such as myocardial infarction or hypertensioninduced pressure overload, respectively. Cardiac failure eventually ensues, with medical intervention typically focused on improving cardiac work capacity or reducing workload. Amelioration of the underlying fibrosis, if it occurs at all, is generally minimal and secondary to improved cardiac function. At first blush, IPF appears starkly different from cardiac fibrosis: an age-related chronic disease of the lung interstitium with a potentially important genetic component and average survival limited to only 3 years [2] . Yet key commonalities in pathophysiology exist between IPF Received: May 9, 2013 Accepted: May 9, 2013 Published online: July 17, 2013

Current and Future Strategies for the Diagnosis and Treatment of Cardiac Fibrosis

Cardiac fibrosis ensues from a mismatch between extracellular matrix production and degradation, resulting in increased and excessive deposition of matrix components including fibrillar collagen types I and III. Increased collagen synthesis and cross-linking strengthens the myocardium but also increases wall stiffness and thereby negatively impact both diastolic (filling) and systolic (contractile) function. Myocardial fibrosis occurs secondary to a host of cardiovascular diseases, including hypertension, coronary artery disease with or without myocardial infarction, myocarditis of various origins, systemic fibrotic diseases (sclerosis), and congenital heart defects (including dilated and hypertrophic cardiomyopathies) (Chaturvedi et al., Circulation 121(8):979–988, 2010). Fibrosis is a significant contributor to the pathogenesis of heart failure (HF), which causes the majority of hospitalizations in patients over 65, and thus represents a considerable but largely ignored clinical concern (Biernacka and Frangogiannis, Aging Dis 2(2):158–173, 2011).